SUMMARY - Protein tyrosine phosphorylation and dephosphorylation, which are balanced by counteracting protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are essential for molecular communications in signal transduction cascades. The receptor-like PTPs (RPTPs) are a family of cell surface PTPs containing a usually large extracellular domain, a single-pass transmembrane domain, and either a single or tandem cytoplasmic phosphatase domain. The functions of RPTPs are attributable to both catalytic activities and extracellular interactions, resembling that of receptor tyrosine kinases (RTKs). However, our molecular understanding of RPTP activity regulation is far from complete compared with that of the structurally and functionally well-characterized RTKs. Structural studies focused on phosphatase domains failed to define generalizable and conclusive mechanisms. We have been engaged in structural and functional analysis of an important RPTP family member, namely CD148/PTPRJ, which is the most abundant RPTP in platelets and megakaryocytes and has an established positive role in platelet aggregation, an essential process for hemostasis and thrombosis. Our preliminary biochemical and structural studies of CD148 have yielded many intriguing observations that begin to define the structural basis of CD148 activity regulation, supporting our hypothesis that dimerization of RPTPs is largely attributable to extracellular and transmembrane domains that govern dimer formation of the phosphatase domain and regulation of catalytic activity. Using innovative construct designs and a novel human megakaryocyte progenitor cell line derived from induced pluripotent stem (iPS) cells, we will examine how the CD148 activity is regulated by dimerization and ligand binding, and how these regulations affect the function of human megakaryocytes and platelets. Using the recently developed proximity-dependent labeling method, we will perform proteomic profiling of CD148 substrates in both resting and activated human megakaryocytes and platelets. Using a combination of crystallographic, electron microscopy, nuclear magnetic resonance, and other multifaceted biochemical and biophysical approaches, we will define the structural basis of CD148 activity regulation through the size of ectodomain and the dimerization mediated by the extracellular and transmembrane domains, and the structural basis of ligand binding. The effect of individual domains, N- linked glycosylation, specific disease-associated polymorphisms, and ligand binding on the structure, dimerization, and activity of CD148 will be examined. These complementary Specific Aims will advance our understanding of the molecular basis for the regulation of CD148 PTP activity and will facilitate the development of novel strategies for modulating CD148 function by selectively targeting either dimerization or ligand binding in the treatment of diseases such as thrombosis and cancer. Approaches established in this study will be readily applicable to other members of the RPTP family, which will improve our family-wide understanding of the molecular mechanisms of RPTP activity regulation.